Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome problems with expression variability, unpredictable impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted. Such systems can also provide approaches to manipulating endogeneous genes. Of course, targeting system requires the ability to focus the recombination process to favor recovery of desired targeting events.
The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Since the primary approaches to gene targeting involve recombinases, whether operating in their natural in vivo environment (as during normal recombination) or as part of schemes that involve pretreatment of substrates so as to associate DNA with a recombinase and increase efficiency of targeting (e.g., double D-loop), there is a continuing need to isolate and characterize the genes for these molecules. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could avoid possible problems of incompatibility associated with molecular interactions due to heterologous components.
Sequences for the bacterial RecA recombinase and functional homologs from yeast and several animal species have been disclosed in various publicly accessible sequence databases. Numerous publications characterizing these recombinases exist (see, e.g., Kowalczykowski et al., Annu. Rev. Biochem., 63:991-1043 (1994)). Reports of the use of bacterial RecA in association with DNA sequences to manipulate homologous target DNA, including improvement of the efficiency of gene targeting in non-plant systems, have been published (see, e.g., PCT published Patent Application Nos. WO 87/01730 and WO 93/22443).
The catalysis of in vitro pairing and strand exchange between circular viral single strand DNA (xe2x80x9css DNAxe2x80x9d) and linear duplex DNA (xe2x80x9cds DNAxe2x80x9d) by a RAD51 recombinase from S. cerevisiae has also been reported (see, e.g., Sung, Science, 265:1241-43 (1994); Kanaar, et al., Nature 391:335-338 (1998); Benson, et al. Nature 391:401-410 (1998)). To date, work with recombinase enzymes in plants, however, has been very limited. Accordingly, there is an ongoing need for the identification and characterization of the functional activities of recombinase enzymes which may offer improved and expanded methods for use in plant systems, particularly agriculturally important crop species such as maize.
Polynucleotide sequences, which encode putatively active RAD51 recombinases, have been isolated from maize. Specifically, cDNA clones ZmRAD51A (SEQ ID NOS: 1) and ZmRAD51B (SEQ ID NOS: 5) from a maize tassel library have been identified and sequenced. The cDNA sequences include 3xe2x80x2-untranslated regions (SEQ ID NOS: 4 and 8) suitable for use in making gene-specific probes, e.g., which can be used to map the locus of the respective ZmRAD51 gene in an RFLP map of a maize population. The RFLP probes are typically at least 15 nucleotide residues, although smaller and larger sizes may also be used. The present invention also includes expression cassettes, vectors, and host cells that incorporate the ZmRAD51 genes. Monocot cells, such as maize cells, are particularly preferred as host cells. In addition, a nuclear localization sequence comprising the 5xe2x80x2 end of the ZmRAD51 gene is identified.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide of at least 10 contiguous amino acids encoded by the isolated nucleic acid of ZmRAD51A or ZmRAD51B. In some embodiments, the polypeptide has a sequence selected from the group consisting of SEQ ID NOS: 3 and 7.
In yet another aspect, the present invention relates to a transgenic plant comprising a expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. Methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention are also included. In some embodiments, the transgenic plant is Zea mays. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of making maize recombinase by transforming or transfecting a host cell with an expression vector containing one of the isolated nucleic acids of the present invention and purifying the recombinase protein from the host cell. In some embodiments, the host cell is a bacterial cell, a yeast cell, or a plant cell.